1. Volume 1, Issue 2, Pages 309-317 (January 1998)
On the Mechanism of RecA-Mediated Repair of Double-Strand Breaks: No Role for Four-Strand DNA Pairing Intermediates 
Qun Shan, Michael M Cox 
Molecular Cell 
Volume 1, Issue 2, Pages (January 1998)
DOI: /S (00)

2. Figure 1
Bypass of Double-Strand Breaks during Four-Strand Exchange Reactions Promoted by RecA Protein
(A) The double-strand break bypass reaction (West and Howard-Flanders 1984). The gapped duplex substrate is depicted linearly, with its circularity indicated with the dashed line. Once the first linear duplex undergoes exchange, the second is presumed to initiate exchange in a region (open arrow) where it must pair with a duplex DNA segment within the filament.
(B) Unwinding of a distal duplex DNA segment after a four-strand exchange reaction. After the reciprocal exchange in step 1, RecA protein will unwind 100 bp or more in a duplex region attached to the region already exchanged, so as to separate the reaction products (MacFarland et al. 1997).
(C) Alternative mechanism for double-strand break bypass, based on the observation in (B). After the exchange of the first linear duplex fragment, continued unwinding of the gapped duplex DNA would open a single-stranded region (open arrow) in which the second linear fragment could initiate DNA strand exchange as a three-stranded reaction.
(D) The presence of a nick in the exchanging strand of the gapped duplex should block double-strand break bypass by the mechanism of (C), but should not block bypass if it proceeds via a four-strand DNA pairing interaction at a point indicated by the open arrow.
Molecular Cell 1998 1, DOI: ( /S (00) )

3. Figure 2 DNA Substrates Used in This Study
All DNA molecules were based on M13mp (8269 bp total). Numbers around the periphery in the molecule at top left are distances, in bp, between the restriction sites indicated. The gapped and nicked gapped DNAs are shown at bottom, and the linear duplex substrates are shown at the upper right. Symbols here and in subsequent figures are: (GD), gapped duplex DNA; (NGD), gapped duplex DNA with a nick in the shortened strand at the AlwNI site; (LDS), linear duplex substrate; (X), (Y), (X′), (Y′), fragments of LDS as shown. Strand shading is maintained in all subsequent figures.
Molecular Cell 1998 1, DOI: ( /S (00) )

4. Figure 3
RecA-Mediated Bypass of a Double-Strand Break during a Four-Strand Exchange Reaction
The individual reactions in the gel are identified by reaction numbers to match the schematic diagram at left. Reaction 1 (at right in the gel) is a normal four-strand exchange. Reaction 2 is the DS break bypass. The bypass is indicated by the formation of both products P1 and P2. Strand exchange reactions in this and subsequent figures were carried out at 37°C and contained 20 mM Tris–HCl (pH 7.5), 25 mM Mg chloride, 2 mM DTT, 2 mM ATP, 0.1 mg/ml BSA, 5% (w/v) glycerol, an ATP regeneration system (8 mM phosphocreatine, 8 units ml−1 phosphocreatine kinase), gapped duplex DNA (9.7 μM), linear duplex (12 μM total), RecA protein (5 μM), and SSB protein (0.15 μM). Symbols are as defined in Figure 2, except that (I) denotes intermediates.
Molecular Cell 1998 1, DOI: ( /S (00) )

5. Figure 4
The Bypass of Double-Strand Breaks Is Abolished by a Nick in the Gapped Duplex DNA Substrate
Reaction 2 is the same bypass reaction shown in Figure 3. Reaction 3 makes use of a gapped duplex with a nick in the exchanging strand coincident with the double-strand break separating X and Y. Both panels show the same gel, with (A) showing a fluoro-image of the gel stained with ethidium bromide, and (B) the same gel monitored with a phosphor-imager. P1′ is the product generated by exchange of the nicked gapped duplex (NGD) with fragment X alone. In reaction 3, P1 (or P1′) is the only new detectable product generated, since P3 and P4 comigrate with the substrates X and Y. A band corresponding to P1 is formed quite early in reaction 3 (A). A 32P label placed on the ends of one strand in each linear substrate (*) allows a discrimination between P1 and P1′, since a label will appear in the P1 band only if fragment Y undergoes exchange.
Molecular Cell 1998 1, DOI: ( /S (00) )

6. Figure 5
The Fragments X and Y Are Exchanged Sequentially Rather Than Concurrently in a Four-Strand Exchange Reaction
The reactions shown here are the same as in Figure 4, except that the fragments X and Y are added at different times. There are four experiments; from left to right in the panels, the first two are reaction 2 and the others are reaction 3, each with six time points. Both panels show the same gel, with (A) and (B) representing a fluoro-image and a phosphor-image, respectively. Both strands of fragment Y are labeled in all cases as indicated for reaction 2. In the first reaction, at left, fragment X is added alone for 60 min. Immediately after the 0 time point, fragment Y is added. In the second reaction, fragment Y is added alone at −60 min. Reaction intermediates and products are generated slowly and only after the addition of X immediately after the 0 time point. In the third and fourth reactions, involving the nicked gapped duplex, a similar reaction protocol is used adding X or Y first, respectively. As seen in (B), there is no incorporation of label into products indicating a reaction with Y to generate either intermediates or products in the third or fourth reactions. Symbols are defined in the legends to Figure 2–Figures 3; 4.
Molecular Cell 1998 1, DOI: ( /S (00) )

7. Figure 6 The Bypass of DS Breaks Requires ATP Hydrolysis
Reactions were carried out as described in the Experimental Procedures and the legend to Figure 3, with dATP replacing ATP. The protocol is similar to the first reaction in Figure 5. Reactions are preincubated for 60 min with fragment X, generating branched strand exchange intermediates. There was then a second short incubation (10 min) in the presence or absence of the RecA mutant K72R (5 μM) protein. Fragment Y is added immediately after the 0 time point in both reactions. Symbols are defined in the legends to Figure 2–Figures 3; 4.
Molecular Cell 1998 1, DOI: ( /S (00) )

8. Figure 7
Scheme for DS Break Bypass Mediated by RecA Protein in the Course of a Four-Strand Exchange Reaction
The gapped duplex is depicted linearly (with the dashed lines in [A] there to indicate that the two ends are actually joined). Fragment X is exchanged first, proceeding to completion (C). At this point, the remaining duplex DNA in the original gapped molecule must be partially unwound (D). This is brought about by the indirect application of DNA torsional stress, since a nick at this position in the exchanging strand blocks the bypass. This could occur by rotating the two hybrid DNA segments, as described previously (MacFarland et al. 1997) and in the inset. The unwinding creates a short single-stranded region where fragment Y can initiate exchange as a three-strand reaction (E). Completion of exchange generates the final products (G).
Molecular Cell 1998 1, DOI: ( /S (00) )